Luminous compounds and labeling reagents using the same

ABSTRACT

This invention provides: a compound represented by formula (I):
 
R—Y—(—X-Phe-COCH 2 COC n F 2n+1 ) m    (I)
 
wherein R denotes hydrogen, alkyl, phenyl, or a group capable of binding to a protein, peptide, amino acid, nucleic acid, or nucleotide; Y denotes CH 2 , a carbocyclic ring, or a heterocyclic ring; X denotes O, S, NH, CH 2 , OCH 2 , CONH, or NHCO; Phe denotes phenylene; n is an integer between 1 and 5; and m is 1, 2, or 3. This inventions also provides a luminous complex of such compound and a rare earth ion, a labeling reagent comprising such compound or luminous complex, and a process for labeling a protein, peptide, amino acid, nucleic acid, or nucleotide using such labeling reagent.

This application is the national stage of PCT/JP02/10511, filed Oct. 10,2002, and published as WO 03/033447 on Apr. 24, 2003.

TECHNICAL FIELD

The present invention relates to a compound for a labeling reagentsuitable for use in time-resolved fluorometry, delayed phosphorimetry,or fluorescence resonance energy transfer assay employed in nucleic acidassay, immunoassay, and chemiluminescence assay. More particularly, thepresent invention relates to a compound that is capable of emittingfluorescence, delayed fluorescence, or phosphorescence when it becomes acomplex through coordinate bonding with metal ions.

BACKGROUND ART

Immunoassays or nucleic acid assays are carried out by a variety ofmeans including visual and radioactivity analyses. Fluorescenceintensities of fluorophores or substances labeled with fluorescent dyesare often measured because of the simplicity of the procedure. Thistechnique is useful because the wavelength of the generated fluorescenceor luminescence is in a range that is different from that of theexcitation light, and can thus be accurately detected. Fluorescence orluminescence, however, disappears in a relatively short period of time,this enables the fluorescence or luminescence to be detected atsubstantially the same time as the generation of the excitation light,so that noises generated by the excitation light are also detected, andthe background noises are sometimes heightened.

Peaks of the fluorescence or emission wavelength often exist inwavelength ranges that are longer than the peaks of the excitationwavelength. Fluorescence or emission is separated from the excitationlight using, for example, a filter for selecting a wavelength and thenits intensity is measured. The difference between the excitationwavelength and the emission or fluorescence wavelength is generallyreferred to as a Stokes shift. In fluorescence assays, a Stokes shift issmall. Thus, it is sometimes difficult to distinguish the excitationlight from the fluorescence emission based on differences inwavelengths.

Fluorescent dyes such as rhodamine and fluoresceine have widefluorescence wavelength distributions, therefore broad fluorescencewaveforms can be obtained. When simultaneous detections of severalsubstances using several fluorophores or fluorescent dyes are intended,it is difficult to assay individual substances due to the overlappingfluorescence wavelength regions. Alternatively, this assay requires theapplication of a conversion formula to analyze the algorithm in order tofind out the amount of each substance.

Chemiluminescence techniques including electrochemiluminescencetechnique and enzymatic chemiluminescence technique are sometimesemployed as techniques in which excitation lights do not interfere withthe emission light. In these techniques, labeled bodies or labeling dyesare not irradiated with the excitation light, and thus, the lightemission only is detected. A technique has been developed in whichisoluminol or acridium ester is labeled to detect the object substance.Another technique involves a highly-sensitive assay system that utilizesreactions between peroxidase and luminol in which the enzyme is employedas a labeling substance and this labeling substance allows a substrateto emit light, or reactions between adamantyl 1,2-dioxetanearylphosphate (AMPPD) and alkali phosphatase.

Fluorescence or phosphorescence of a complex comprising a rare earthelement, such as europium or samarium, and a ligand thereof has a longlasting light emission. Recently, time-resolved fluorometry orphosphorimetry that makes use of this advantage has been developed as anexcellent technique. Properties, such as a long period of fluorescenceor phosphorescence being quenched, a large Stokes shift, and sharp peakwaveforms of fluorescence and phosphorescence, enable the detection offluorescent or phosphorescent signals while excluding noises caused bythe excitation light. Thus, a highly sensitive analytical technique canbe provided. In this technique, the excitation light is applied via axenon flash lamp or laser beam while pulsing, and the fluorescence orphosphorescence of interest is detected after the fluorescence of theapparatus or other substances caused by the excitation lightsdisappears. This ligand is improved, and compounds such as4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid(BCPDA) or4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)chlorosulfo-o-terphenyl(BHHCT) have been reported. BHHCT is disclosed in, for example,JP-A-09-241233. These are excellent chelate compounds, although they arenot yet improved enough to yield the minimal detection sensitivityrequired for immunoassays or nucleic acid assays.

DISCLOSURE OF THE INVENTION

The present invention relates to labeling reagents suitable for use intime-resolved fluorometry, delayed phosphorimetry, or fluorescenceresonance energy transfer assay employed in nucleic acid assay,immunoassay, and chemiluminescence assay. An object of the presentinvention is to provide a useful labeling reagent that can detect asubstance of interest in a sample with high sensitivity and a compoundsuitably used in a labeling reagent.

The present invention includes the following inventions.

(1) A compound represented by formula (I):R—Y—(—X-Phe-COCH₂COC_(n)F_(2n+1))_(m)  (I)wherein R denotes hydrogen, alkyl, phenyl, or a group capable of bindingto a protein, peptide, amino acid, nucleic acid, or nucleotide; Ydenotes CH₂, a carbocyclic ring, or a heterocyclic ring; X denotes O, S,NH, CH₂, OCH₂, CONH, or NHCO; Phe denotes phenylene; n is an integerbetween 1 and 5; and m is 1, 2, or 3.

(2) The compound according to (1) above represented by formula (1):

wherein * denotes C_(n)F_(2n+1) (wherein n is an integer between 1 and5) and R is as defined in (1) above.

(3) The compound according to (1) above represented by formula (2):

wherein * denotes C_(n)F_(2n+1) (wherein n is an integer between 1 and5) and R is as defined in (1) above.

(4) The compound according to (1) above represented by formula (3):

wherein * denotes C_(n)F_(2n+1) (wherein is an integer between 1 and 5)and R is as defined in (1) above.

(5) The compound according to (1) above represented by formula (4):

wherein * denotes C_(n)F_(2n+1) (wherein n is an integer between 1 and5) and R is as defined in (1) above.

(6) The compound according to (1) above represented by formula (5):

wherein * denotes C_(n)F_(2n+1) (wherein n is an integer between 1 and5) and R is as defined in (1) above.

(7) The compound according to (1) above represented by formula (6):

wherein * denotes C_(n)F_(2n+1) (wherein n is an integer between 1 and5) and R is as defined in (1) above.

(8) The compound according to (1) above represented by formula (7):

wherein * denotes C_(n)F_(2n+1) (wherein n is an integer between 1 and5) and R is as defined in (1) above.

(9) The compound according to (1) above represented by formula (8):

wherein * denotes C_(n)F_(2n+1) (wherein n is an integer between 1 and5) and R is as defined in (1) above.

(10) The compound according to (1) above represented by formula (9):

wherein * denotes C_(n)F_(2n+1) (wherein n is an integer between 1 and5) and R is as defined in (1) above.

(11) The compound according to (1) above represented by formula (10):

wherein * denotes C_(n)F_(2n+1) (wherein n is an integer between 1 and5) and R is as defined in (1) above.

(12) The compound according to (1) above represented by formula (11):

wherein * denotes C_(n)F_(2n+1) (wherein n is an integer between 1 and5) and R is as defined in (1) above.

(13) The compound according to (1) above represented by formula (12):

wherein * denotes C_(n)F_(2n+1) (wherein n is an integer between 1 and5) and R is as defined in (1) above.

(14) The compound according to (1) above represented by formula (13):

wherein * denotes C_(n)F_(2n+1) (wherein n is an integer between 1 and5) and R is as defined in (1) above.

(15) The compound according to (1) above represented by formula (14):

wherein * denotes C_(n)F_(2n+1) (wherein n is an integer between 1 and5) and R is as defined in (1) above.

(16) The compound according to any of (1) to (15) above, wherein Rdenotes a group in which a functional group capable of binding to aprotein, peptide, amino acid, nucleic acid, or nucleotide is bound to aterminus of a spacer comprising a carbon chain that may optionallycontain, as a member of this chain, at least one hetero atom selectedfrom among oxygen, nitrogen, and sulfur.

(17) The compound according to any one of (1) to (16) above, wherein Rcomprises at least one functional group represented by the followingformulae:

wherein X is selected from among a halide atom, —OSO₃CH₃, —OSO₂F,—OSO₂CF₃, —SO₂C₄F₉, and

R^(A) is selected from among alkyl, alkenyl, aryl, and aralkyl; R^(B) isselected from among alkylene, arylene, and aralkylene; p is an integerbetween 0 and 5, inclusive; q is an integer between 2 and 10, inclusive;and n is an integer between 1 and 20, inclusive.

(18) A fluorescent complex comprising the compound according to any oneof (1) to (17) above and a rare earth ion.

(19) A labeling reagent comprising the compound according to any one of(1) to (17) above or the fluorescent complex according to (18) above.

(20) The labeling reagent according to (19) above for immunoassays ornucleic acid assays.

(21) A method for labeling a protein, peptide, amino acid, nucleic acid,or nucleotide utilizing the labeling reagent according to (19) or (20)above.

In formula (I), R denotes hydrogen, alkyl, phenyl, or a group capable ofbinding to a protein, peptide, amino acid, nucleic acid, or nucleotide.The “group capable of binding to a protein, peptide, amino acid, nucleicacid, or nucleotide” used herein is not particularly limited as long asit is capable of binding to any of the amino, carboxyl, mercapto, orhydroxyl group existing in a target protein, peptide, amino acid,nucleic acid, or nucleotide. Examples thereof include groups having atleast one functional group represented by the following formulae:

wherein X is selected from among a halide atom, —OSO₃CH₃, —OSO₂F,—OSO₂CF₃, —SO₂C₄F₉, and

R^(A) is selected from among alkyl, alkenyl, aryl, and aralkyl; R^(B) isselected from among alkylene, arylene, and aralkylene; p is an integerbetween 0 and 5, inclusive; q is an integer between 2 and 10, inclusive;and n is an integer between 1 and 20, inclusive.

In the above formulae, a halide atom (halogen atom) denoted by X iseither fluoride (fluorine), chloride (chlorine), bromide (bromine), oriodide (iodine). Concerning groups denoted by R^(A), for example, alkylis C₁₋₆ alkyl (e.g., methyl or ethyl), alkenyl is C₂₋₆ alkenyl (e.g.,vinyl or propenyl), aryl is phenyl or naphthyl, and aralkyl is benzyl,phenethyl, or naphthylmethyl. Concerning groups denoted by R^(B), forexample, alkylene is C₁₋₆ alkylene (e.g., —(CH₂)_(n)— wherein n is aninteger between 1 and 6), arylene is phenylene or naphthylene, andaralkylene is —Ar—(CH₂)_(n)— wherein Ar is phenylene or naphthylene andn is an integer between 1 and 6.

When R is a group capable of binding to a protein, peptide, amino acid,nucleic acid, or nucleotide, the protein, peptide, amino acid, nucleicacid, or nucleotide to which R is bound is not particularly limited.Examples thereof include antibodies, labeled antibodies, antigens,biotin, avidin, streptavidin, bovine serum albumin, hapten, hormone,polypeptide, and polynucleotide.

Examples of carbocyclic rings denoted by Y include an optionallysubstituted aromatic monocyclic hydrocarbon (e.g., a benzene ring), a 3-to 8-membered ring comprising an optionally-substituted saturatedmonocyclic hydrocarbon (e.g., a cyclohexane ring), a 4- to 8-memberedring comprising an optionally substituted unsaturated monocyclichydrocarbon (e.g., a cyclohexene or cyclopentadiene ring), an optionallysubstituted fused polycyclic hydrocarbon (e.g., a naphthalene,phenanthrene, or fluorene ring), and a hydrocarbon ring complex (e.g., abiphenyl, terphenyl, phenanthrene, or fluorene ring).

Examples of heterocyclic rings denoted by Y include an optionallysubstituted aromatic heterocyclic ring (e.g., a thiophene ring), anoptionally substituted 3- to 8-membered saturated heterocyclic ring(e.g., a tetrahydrofuran ring), an optionally unsaturated heterocyclicring (a pyroline ring), and an optionally substituted fused heterocyclicring (e.g., a benzothiophen, dibenzothiophene, benzofuran, ordibenzofuran ring).

Phenylene denoted by Phe is preferably 1,4-phenylene.

The rare earth ion used in the present invention is preferably alanthanoid ion, and examples thereof include europium (Eu), samarium(Sm), terbium (Tb), and dysprosium (Dy) ions.

The molecular size of a chelate compound comprising a metal element isrelatively small. A peptide or amino acid, particularly a protein ornucleotide chain, labeled via a chain (spacer) mainly composed of carbon(C) may be more advantageous. A protein or nucleotide chain often has acomplicated three-dimensional structure, and a binding site of a labelwith a functional group sometimes exists inside this structure. Use of aspacer is effective when these substances are intended to be labeled.

An example of the aforementioned spacer is a spacer comprising a carbonchain that may optionally contain, as a member of this chain, at leastone hetero atom selected from among oxygen, nitrogen, and sulfur.

A specific example of R comprising the aforementioned spacer isrepresented by the formula:—(CH₂)_(a)—FG or—[(CH₂)_(b)—A]_(c)—(CH₂)_(d)—FGwherein a is an integer between 1 and 40; b is an integer between 1 and20; c is an integer between 1 and 10; d is an integer between 0 and 20;A denotes oxygen (O), nitrogen (NH), or sulfur (S); and FG denotes afunctional group.

Fluorescence is generated at the time of transition from the firstexcited state to the ground state, and phosphorescence is generated atthe time of transition from one state to another state having differentspin multiplicity. In the past, a labeling reagent having a β-diketonestructure and a labeling reagent having an aromatic group existed ascomplex reagents coordinately bonded to heavy metal ions. Fluoresceinbridged with —O— (oxygen) exhibits intensive fluorescence orphosphorescence whereas emission of fluorescence or phosphorescence isnot observed in the case of phenolphthalein. Quinoline to which abenzene ring had been applied exhibits fluorescence or phosphorescencewhile no fluorescence or phosphorescence is observed in the case ofpyridine.

When the intensity of fluorescence or phosphorescence is intended to beenhanced, a spacer that has both an aromatic group and O (oxygen), thatis stable, and that yields high efficiency of synthesis isadvantageously used.

In immunoassays or nucleic acid assays, a solid phase is utilized as aplatform for reaction, upon which a product of antigen-antibody reactionor a product of DNA or RNA hybridization is formed, and the resultant isthen assayed. A label or labeling dye is first bound to an antigen,antibody, DNA, or RNA to compose the reaction product. Alternatively, itis allowed to bind to a reaction product at the final stage with theutilization of, for example, reactions between avidin and biotin, andmeasurement is then initiated.

The compound and the labeling reagent of the present invention have aβ-diketone structure and an aromatic group. They are comprised of O, S,NH, CH₂, OCH₂, CONH, or NHCO denoted by X between phenylene denoted byPhe and CH₂, a carbocyclic ring, or a heterocyclic ring denoted by Y.This construction enlarges the range of motion of the compound and thelabeling reagent at the β-diketone site for bridging rare earthelements, exemplified by europium, and rare earth ions can be moreeffectively sustained. Thus, rare earth ions are more steadilycoordinately bound with the β-diketone site. Accordingly, fluorescenceor phosphorescence can be certainly emitted when irradiated withadequate excitation light.

The compound and the labeling reagent of the present invention arecomprised of O, S, NH, CH₂, OCH₂, CONH, or NHCO denoted by X betweenphenylene denoted by Phe and a carbocyclic or heterocyclic ring denotedby Y as mentioned above. This construction allows an aromatic group(phenylene group) to be located discontinuously from a carbocyclic orheterocyclic ring. Thus, an effective chelate compound can be obtainedwithout significantly inhibiting hydrophilic properties.

The compound and the labeling reagent of the present invention arecomprised of O, S, NH, CH₂, OCH₂, CONH, or NHCO denoted by X betweenphenylene denoted by Phe and a carbocyclic or heterocyclic ring denotedby Y. Because of this construction, the compound and the labelingreagent of the present invention are less likely to be affected by theelectronic absorption effects at sites where they counter C_(n)F_(2n+1)(wherein n is an integer between 1 and 5) and the β-diketone site. Thisfacilitates the introduction of reactive groups. Accordingly, conjugatesthereof with an amino acid, peptide, protein, or nucleic acid can bemore efficiently obtained.

The compound and the labeling reagent of the present invention arecomprised of O, S, NH, CH₂, OCH₂, CONH, or NHCO denoted by X betweenphenylene denoted by Phe and a carbocyclic or heterocyclic ring denotedby Y. Because of this construction, when the compound and the labelingreagent of the present invention are allowed to bind to solid phasecarriers such as amino acids, peptides, proteins, nucleic acids, orplastic particles via a reactive group, the compound and the labelingreagent become less likely to be affected by these substances at theβ-diketone site and they can effectively sustain rare earth ions. Thus,rare earth ions can be more steadily coordinately bound with theβ-diketone site. This enables the emission of certain fluorescence orphosphorescence when irradiated with adequate excitation light.

Further, when the compound and the labeling reagent of the presentinvention employ a spacer in R, the functional group of R becomes morelikely to bind to a protein, nucleotide chain, or the like. This canincrease the number of labels per molecule of the labeled substance.

The compound of the present invention can be produced, for example, inthe following manner.

(Production Process 1)

The First StepR—Y—(—X-Phe-H)_(m)+CH₃COX==>R—Y—(—X-Phe-COCH₃)_(m)wherein X denotes a halogen atom, such as chlorine, and R, Y, X, and Pheare as defined in (I) above.

This reaction is so-called Friedel-Crafts acylation and it can becarried out in accordance with a conventional technique.Dichloromethane, chloroform, or the like is used as a solvent, andreaction is carried out in the presence of a Lewis acid such as aluminumchloride.

The Second StepR—Y—(—X-Phe-COCH₃)_(m)+C_(n)F_(2n+1)COOR¹==>R—Y—(—X-Phe-COCH₂COC_(n)F_(2n+1))_(m)wherein R¹ denotes lower alkyl, such as methyl or ethyl, and R, Y, X,Phe, n, and m are as defined in (I) above.

This reaction is the condensation of a ketone compound and an estercompound, and it can be carried out in accordance with any conventionaltechnique. Cyclohexane, n-hexane, diethyl ether, or the like is used asa solvent, and reaction is carried out in the presence of sodiumhydride, metalalkoxide, or the like.

(Production Process 2)

This production process is employed when X denotes CH₂.

The First StepR—Y—(—CH₂—Br)_(m)+(HO)₂B-Phe-COCH₃==>R—Y—(—CH₂-Phe-COCH₃)_(m)wherein R, Y, Phe, and m are as defined in (I) above.

This reaction is carried out in the presence ofPdCl₂(dppf).CH₂Cl₂(dppf=1,1′-bis(diphenylphosphino)ferrocene) in asolvent such as a mixed solvent of tetrahydrofuran and water.

The Second StepR—Y—(—CH₂-Phe-COCH₃)_(m)+C_(n)F_(2n+1)COOR¹==>R—Y—(—CH₂-Phe-COCH₂COC_(n)F_(2n+1))_(m)wherein R¹ denotes lower alkyl, such as methyl or ethyl, and R, Y, Phe,n, and m are as defined in (I) above.

This reaction can be carried out in the same manner as with the secondstep of Production Process 1.

(Production Process 3)

This production process is employed when X denotes O.

The First StepR—Y—(—OH)_(m)+F-Phe-COCH₃ (or Br-Phe-COCH₃)==>R—Y—(—O-Phe-COCH₃)_(m)wherein R, Y, Phe, and m are as defined in (I) above.

This reaction is carried out by adding K₂CO₃, NaOH, NaH, or the like toa solvent.

The Second StepR—Y—(—O-Phe-COCH₃)_(m)+C_(n)F_(2n+1)COOR¹==>R—Y—(—O-Phe-COCH₂COC_(n)F_(2n+1))_(m)wherein R¹ denotes lower alkyl, such as methyl or ethyl, and R, Y, Phe,n, and m are as defined in (I) above.

This reaction can be carried out in the same manner as with the secondstep of Production Process 1.

The thus obtained compounds can be used as labeling reagents forproteins, peptide, amino acids, nucleic acids, or nucleotides.Alternatively, they can be used by being immobilized onto the surfacesof carriers such as plastic particles or being included in hollow bodiessuch as liposomes.

Labeling reactions utilizing the compound of the present invention canbe carried out by selecting suitable reactions in accordance with thecorrelation between the functional group in the compound and afunctional group in a protein or nucleic acid. For example, labeling canbe carried out by amide formation between chlorosulfonyl or carboxyl inthe compound of the present invention and an amino group in the protein.This amide formation easily proceeds in a carbonate buffer or Tris-HClbuffer (pH: between 9.0 and 9.5) at room temperature.

Examples of immunoassays utilizing the labeling reagent of the presentinvention are time-resolved fluoroimmunoassays and specific bindingassays utilizing antigen-antibody reactions. The term “time-resolvedfluoroimmunoassays” refers to highly sensitive fluoroimmunoassays inwhich only the fluorescent signals of the label are subjected totime-resolved fluoroassays using a long-lived fluorescent label (e.g.,Eu-chelate) after the short-lived background fluorescence disappeared.The term “specific binding assays” refers to, for example, immunoassaysutilizing antigen-antibody reactions, assays utilizing areceptor-acceptor bond, or assays utilizing nucleic acid hybridization.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of gel chromatography analysis of Compound (b)obtained in Example 1.

FIG. 2 shows the results of HPLC analysis of Compound (b) obtained inExample 2.

FIG. 3 shows the results of NMR spectrum analysis of Compound (b)obtained in Example 2.

FIG. 4 shows the results of TOF/MS spectrum analysis of Compound (b)obtained in Example 2.

FIG. 5 shows the compounds of the present invention used in examples 5to 7 and other β-diketone (1,3-dione) compounds.

FIG. 6 is a table and a graph showing: the fluorescent signals obtainedfor the compounds of the present invention with the use of an apparatusfor time-resolved assay; the maximal excitation wavelengths for theexcitation spectra; and the maximal absorption wavelengths for theabsorption spectra.

FIG. 7 shows the fluorescence decay curves for the compounds of thepresent invention.

FIG. 8 shows the calibration curves for α-fetoprotein (AFP) obtained byusing the compounds of the present invention as labels.

FIG. 9 shows the distinctive features of the present invention withreference to an embodiment of the compound of the present invention.

FIG. 10 shows the calibration curves for the europium-labeled bovineserum albumin (BSA) according to the present invention.

FIG. 11 shows the assay results for human C-reactive proteins (CRP)according to the present invention.

This description includes part or all of the contents as disclosed inthe description and/or drawings of Japanese Patent Application No.2001-312562, which is a priority document of the present application.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention is hereafter described in detail with reference tothe following examples, although the technical scope of the presentinvention is not limited to these examples.

EXAMPLE 1

The following is an embodiment of a process for synthesizing a compoundrepresented by formula (6).

The First Step

The following is the first step.

Materials

-   (1) Anhydrous aluminum chloride: 2.18 g-   (2) Anhydrous dichloromethane: 35 ml-   (3) Acetyl chloride: 1.29 g-   (4) Diphenylmethane: 2.5 g    Process of Synthesis

Materials (1) and (2) were added to a flask, and the content therein wascooled to 0° C. Material (3) was further added thereto, 15 ml of adichloromethane solution of material (4) was slowly added dropwise undercooling, and the mixture was stirred at room temperature for 2 hours.Ice (approximately 15 g) was added to the mixture, 20 ml of 1N HClsolution was further added, the precipitated aluminum oxide was allowedto dissolve, and an organic layer was fractionated. An aqueous layer wasextracted three times with 20 ml of dichloromethane. The organic layerwas mixed and rinsed. Anhydrous magnesium sulfate was added, a solventwas removed by distillation under reduced pressure, and a residue waspurified by silica gel chromatography (solvent: n-hexane:ethylacetate=3:2) to obtain Compound (a) (yield: 96%).

The Second Step

The following is the second step.

Materials

-   (1) Sodium hydride (60% in oil): 60 mg-   (2) Anhydrous cyclohexane: 5 ml-   (3) C₃F₇COOC₂H₅: 460 mg-   (4) Compound (a): 200 mg    Process of Synthesis

Materials (1) and (2) were added to a flask, materials (3) and 3 ml of acyclohexane solution (material (4)) were added thereto while heating andstirring, and the mixture was further stirred at room temperature for 30minutes. An aqueous solution of 15% acetic acid was added to themixture, and the resultant was added to 10 ml of ice water. An organiclayer was fractionated with the aid of a separatory funnel, and anaqueous layer was extracted three times with 20 ml of diethyl ether. Anorganic layer was mixed, rinsed, and dried over anhydrous magnesiumsulfate. A solvent was removed by distillation under reduced pressure,and a residue was purified by silica gel column chromatography (solvent:n-hexane:ethyl acetate=3:2) to obtain light yellow Compound (b) (yield:99%).

FIG. 1 shows the results of gel chromatography analysis of Compound (b).The m/z value of Compound (b) was found to be 406 as a result of TOF/MFspectrum analysis.

EXAMPLE 2

The following is an embodiment of a process for synthesizing a compoundrepresented by formula (2).

The First Step

The following is the first step.

Materials

-   (1) 1,2-Bis(bromomethyl)benzene: 5.0 g-   (2) 4-Acetylphenylboronic acid: 13.6 g-   (3) A mixed solution of tetrahydrofuran (THF, 50 ml) and distilled    water (5 ml)-   (4) Cesium carbonate: 18.5 g-   (5) PdCl₂(dppf).CH₂Cl₂: 1.5 g    -   dppf=1,1′-Bis(diphenylphosphino)ferrocene-   Reaction temperature: 70° C. (reflux)-   Reaction period: 1 day    Process of Synthesis

Materials (1), (2), (3), and (4) were mixed in a reaction vessel, andthe resultant was stirred while heating at 70° C. Material (5) was addedthereto 30 minutes later. Heating was terminated 24 hours later, and thereaction solution was added to 60 ml of distilled water, followed byextraction with 80 ml of chloroform. The organic layer was washed with40 ml of an aqueous solution of 5% hydrochloric acid and 40 ml ofdistilled water in that order and then dried over magnesium sulfate. Thesolvent was removed by distillation under reduced pressure, and 6 g of acrude product was obtained. After the product was separated and purifiedby silica gel chromatography, it was further fractionated and purifiedby gel chromatography. Thus, 1.0 g of Compound (a) was obtained (HPLCpurity: 99%, yield: 15%).

¹H NMR (300 MHz, CDCl₃) δ 2.56 (6H, s), 3.96 (4H, s), 7.10–7.15 (6H, m),7.23–7.27 (2H, m), 7.81–7.84 (4H, m)

MS (MALDI TOF) m/z 344 (M+H⁺)

The Second Step

The following is the second step.

Materials

-   (1) Compound (a): 300 mg-   (2) C₃F₇COOC₂H₅: 440 mg-   (3) Anhydrous diether ether: 12 ml-   (4) Sodium methoxide: 99 mg-   Reaction temperature: room temperature-   Reaction period: 1 day    Process of Synthesis

Materials (1), (2), (3), and (4) were mixed in a reaction vessel. Anaqueous solution of 10% sulfuric acid (12 ml) was added thereto 24 hourslater. After the mixture was stirred for 15 minutes, the organic layerwas removed by distillation under reduced pressure, and the precipitatedcrystal was filtered. After the crystal was thoroughly rinsed, it wasadded to 10 ml of ethanol, followed by stirring while heating. Ethanolwas removed by distillation under reduced pressure until the volumethereof became approximately 5 ml, 20 ml of petroleum ether was addedthereto, and the mixture was stirred while heating. Insoluble matterswere filtered, and the solvent was removed by distillation. Thus, 300 mgof Compound (b) was obtained. The compound was fractionated and purifiedby gel chromatography to obtain 100 mg of Compound (b).

MS (MALDI TOF) m/z 735 (M+H⁺)

FIG. 2 shows the results of HPLC analysis of Compound (b), FIG. 3 showsthe results of NMR analysis thereof, and FIG. 4 shows the results ofTOF/MS spectrum analysis thereof.

EXAMPLE 3

The following is an embodiment of a process for synthesizing a compoundrepresented by formula (1).

The First Step

The following is the first step.

Materials

-   (1) 1,2-Dihydroxybenzene-   (2) 4′-Fluoroacetophenone    Process of Synthesis

Materials (1) and (2) were allowed to react with each other in thepresence of potassium carbonate dissolved in a solvent, i.e.,N,N-dimethylacetamide, to obtain Compound (a).

The Second Step

The following is the second step.

Materials

-   (1) Compound (a): 1.75 g-   (2) Anhydrous diethyl ether: 40 ml-   (3) Sodium methoxide: 1.36 g-   (4) C₃F₇COOC₂H₅: 3.67 g    Process of Synthesis

Materials (1) and (2) were mixed together, the mixture was allowed tocool, and material (3) was added thereto. Further, 10 ml of a diethylether solution of material (4) was added dropwise thereto. The mixturewas allowed to cool and stirred for 1 hour. Diethyl ether was addedthereto, the pH level was adjusted to 4 with the aid of dilutehydrochloric acid, and the resultant was washed with water and saturatedsaline in that order. The resultant was dried over anhydrous sodiumsulfate, and the solvent was removed by distillation under reducedpressure. Thus, 3.87 g of reddish-brown paste was obtained. This pastewas purified by wet column chromatography (solvent: hexane/ethylacetate=4/1), and 2.69 g of reddish-brown paste was obtained.

EXAMPLE 4

A luminous compound comprising the compound of the present invention wasprepared. TTA (4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione) and BFA(4,4,4-trifluoro-1-phenyl-1,3-butanedione) were purchased from DojindoLaboratories. Synthesis of BHHT (4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)-o-terphenyl) wascarried out in reference to JP-A-09-241233 and Yuan and Matsumoto(Analytical Chemistry, 1998, 70, pp. 596–601).

FIG. 5 shows structural formulae of these compounds.

EXAMPLE 5

A compound shown in FIG. 5 comprising the compound of the presentinvention was dissolved in acetonitrile (Wako Pure Chemical Industries,Ltd.) to a concentration of 10⁻⁴ mol/ml or 10⁻⁵ mol/ml, and the solutionwas further diluted to 10⁻¹⁰ mol/ml by 10-fold serial dilution. Asolution of this compound (10⁻⁷ mol/ml) was subjected to absorptionspectrum analysis using a spectrophotometer U-3300 (HitachiHigh-Technologies). Further, a solution of the aforementioned compound(10⁻¹⁰ mol/ml) was mixed with an aqueous solution comprising 0.2 mMeuropium chloride hexahydrate (EuCl₃.6H₂O, Wako Pure ChemicalIndustries, Ltd.), 0.2 mM TOPO (tri-n-octylphophine oxide, DojindoLaboratories), and 1% Triton X-100 (Sigma) at the mixing ratio of 1:9,and the resulting mixture was incubated at 42° C. for 2 hours. 0.1 ml ofthe incubated solution was dispensed to wells of a 96-well flat bottommicrotiter plate (Nunc), the plate was irradiated with excitation lightat approximately 340 nm, and fluorescence at approximately 615 nm, whichhad been emitted 0.2 msec to 0.8 msec after the irradiation with theexcitation light, was measured using a time-resolved assay apparatus(Hitachi High-Technologies). Further, a similar solution was subjectedto the excitation spectrum analysis using a fluorescence spectrometerF-4010 (Hitachi High-Technologies).

FIG. 6 shows the results of these analyses. Compound 4 was found to beuseful as a fluorophore because it exhibited the highest signalintensity by the assay conducted using a time-resolved assay apparatusand the peak intensity thereof was the highest as a result of thefluorescence spectrum analysis and the absorption spectrum analysis.Compound 6 comprises CH₂ denoted by X between phenylene and acarbocyclic ring denoted by Y. This makes the formation of a conjugatedsystem involving 3 carbocyclic rings difficult. Accordingly, signalsseem to be increased with the number of compound 1 structures.

EXAMPLE 6

The solution of the compound obtained in Example 5 (0.1 ml, 10⁻¹¹mol/ml) was dispensed to wells of a microtiter plate, the plate wasirradiated with excitation light at approximately 340 nm, assay wasinitiated using a time-resolved assay apparatus 0.1, 0.2, 0.3 . . . 0.8,0.9, and 1.0 msec after the irradiation of the excitation light, thevalues representing the emitted signals observed over a period of 0.1msec following the initiation were dotted in the drawing, and these dotswere connected with a line.

FIG. 7 shows the results thereof. The compounds of the present inventionwere found to be capable of maintaining fluorescence for a sufficientlylong period of time. Fluorescent signals exhibited the longesthalf-lives particularly as to Compound 4.

EXAMPLE 7

Assay of α-fetoprotein (AFP)

Assay was carried out according to Yuan and Matsumoto (AnalyticalChemistry, 1998, 70, pp. 596–601).

(1) Labeling of AFP Antibody with Biotin (in Accordance with theProducer's Manual for Sulfo-NHS-LC-biotin, Pierce)

An anti-human AFP antibody (1 mg, DACO Immunoglobulins) was dissolved in1 ml of phosphate buffer saline (PBS, pH, 7.4). Sulfo-NHS-LC-biotin(0.062 mg) was added thereto, and the mixture was allowed to stand in anice bath for 2 hours. Thereafter, a PD-10 column (Pharmacia) was used toelute and collect an antibody fraction with the aid of PBS, and unboundSulfo-NHS-LC-biotin was removed. Sodium azide was added to thebiotin-labeled antibody solution to account for 0.1% of the solution,and the resultant was stored at 4° C.

(2) Introduction of Chlorosulfonyl into the Compound of Example 4

Chlorosulfuric acid (Wako Pure Chemical Industries, Ltd.) was added to acompound shown in FIG. 5 in an amount of 0.2 ml relative to 0.1 mmol ofthe compound shown in FIG. 5. The mixture was stirred at roomtemperature for 7 hours, and the reaction solution was added dropwise to4 ml of pure water (in an ice bath) while stirring. The resultingprecipitate was centrifuged and washed three times with pure water.Thereafter, the precipitate was vacuum dried for 45 hours.

(3) Labeling of a Compound with Streptavidin

Streptavidin (SA, 10⁻⁵ mmol, Chemicon International) was dissolved in 4ml of 0.1M carbonate buffer (pH 9.1). The compound mentioned in (2)above (10⁻³ mmol) was dissolved in 40 μl of ethanol, and the resultantwas added dropwise to a solution of SA. The mixed solution was stirredat room temperature for 1 hour and then thoroughly dialyzed with anaqueous solution of 0.1 M NaHCO₃ containing 0.05% sodium azide. Afterthe dialysis, the pH level was adjusted to 6.8 with the aid of 1M HCl,the total amount was brought to 6 ml, and BSA was added thereto toaccount for 0.1% of the mixture. The resultant was diluted 300-fold witha 0.05M Tris-HCl buffer (pH 7.8) containing 10⁻⁷ M EuCl₃.6H₂O, 1% BSA,and 0.1% sodium azide, heated at 56° C. for 2 hours, and then subjectedto the reaction.

(4) Coating of a Microtiter Plate with an Anti-AFP Antibody

An anti-human AFP antibody (100 μl, Nippon Biotest Laboratories Inc.)diluted to 5 μg/ml with the aid of 0.1M carbonate buffer (pH 9.6) wasdispensed to the wells of a microtiter plate, and the plate was allowedto stand at 4° C. overnight for coating. Thereafter, the plate waswashed with 0.05% Tween 20-containing physiological saline, 100 μl ofcarbonate buffer (pH 9.1) containing 1% BSA and 2% sucrose was added,and the resultant was allowed to stand at 37° C. The plate was washedwith 0.05% Tween 20-containing physiological saline (Sigma) 1 hour laterand then stored at −20° C.

(5) Implementation of Immunoassays

A human AFP standard preparation (DACO Immunoglobulins) was subjected toa 10-fold serial dilution with PBS containing 1% BSA, and 50 μl thereofwas added to each well of a microtiter plate. The plate was shaken at37° C. for 1 hour and then washed with 0.05% Tween 20-containingphysiological saline. Thereafter, the biotin-labeled anti-AFP antibodyobtained in (1) was diluted to 1 μg/ml with the aid of 1% BSA-containingphysiological saline, and 50 μl thereof was dispensed to each well. Theplate was shaken at 37° C. for 1 hour and then washed with 0.05% Tween20-containing physiological saline. Thereafter, 50 μl of thestreptavidin-labeled compound described in the above (3) was dispensedto each well.

The plate was allowed to stand at room temperature for 30 minutes andthen washed with 0.05% Tween 20-containing physiological saline. Themicrotiter plate was subjected to the analysis of the amount of emittedlight using a time-resolved assay apparatus.

FIG. 8 shows the results thereof. As is apparent from the drawing, thecompounds of the present invention can be efficiently used inimmunoassays. In the case of Compound 4, a good calibration curve wasproduced, and the minimal detection sensitivity attained based thereonwas the best value among other compounds, which had been simultaneouslyexamined. A relatively good calibration curve and minimal detectionsensitivity were obtained in the case of Compound 6. Although thiscompound did not exhibit large signals in Example 6, this compound had agood binding property of a reactive group (in this case, chlorosulfonylresidue) and a highly stable trapping property for europium ions at theβ-diketone site. Thus, relatively good results were obtained inantigen-antibody reactions.

In the case of Compound 1, TTA, and BFA having a carbocyclic orheterocyclic ring, reactive groups (in this case, chlorosulfonylresidues) did not bind, nor were the europium ions existing in theβ-diketone site stably sustained. Thus, no signal indicatingantigen-antibody reactions was obtained.

EXAMPLE 8

An embodiment of the compounds of the present invention, i.e., Compound4 shown in FIG. 5, is examined.

Placement of O denoted by X between phenylene and a carbocyclic ringdenoted by Y enlarges the distance between the C₃F₇ or β-diketone siteand the R site. This makes the compound less susceptible to theelectronic absorption effects at the C₃F₇ and β-diketone sites andfacilitates the introduction of reactive groups. Accordingly, conjugatesthereof with an amino acid, peptide, protein, or nucleic acid can bemore efficiently obtained.

Similarly, when the compound is bound to solid phase carriers such asamino acids, peptides, proteins, nucleic acids, or plastic particles viaa reactive group through placement of O denoted by X, the compoundbecomes less susceptible thereto at the β-diketone site. This enablesrare earth ions to be sustained more effectively and stably. Thus, rareearth ions can form better coordinate bonds with the β-diketone site,which assures the emission of fluorescence or phosphorescence whenirradiated with adequate excitation light.

Further, placement of O denoted by X enables more flexible placement ofa ligand at the C₃F₇ site, the β-diketone site, and the carbocyclic ringsubsequent thereto, thereby forming a more stable complex with rareearth ions.

EXAMPLE 9

Compound 4 shown in FIG. 5 was labeled with BSA. Chlorosulfonyl wasintroduced in accordance with the process described in (2) of Example 7.Labeling was carried out according to Yuan and Matsumoto (AnalyticalChemistry, 1998, 70, pp. 596–601).

A solution of DMF (N,N-dimethylformamide, 0.08 ml) containing 1.5 mg ofCompound 4 to which chlorosulfonyl had been introduced was added to 0.4ml of 0.1M carbonate buffer (pH 9.3) containing 2 mg of BSA whilestirring. After the mixture was stirred at room temperature, a BSAfraction labeled with the compound was sampled using a PD-10 column. Inthis case, an aqueous solution of 0.05M NH₄HCO₃ (pH 8.0) was used as aneluate.

The sampled fraction was diluted with a 0.05M Tris-HCl buffer (pH 7.8)containing 10⁻⁷M EuCl₃.6H₂O, 1% BSA, and 0.1% sodium azide, and thediluted product was heated at 56° C. for 2 hours.

Europium-labeled BSA was subjected to a 10-fold serial dilution with theaid of a 0.1M Tris-HCl buffer (pH 9.1) containing 0.05% Tween 20 and0.05% sodium azide, and the amount of emitted light was measured with atime-resolved assay apparatus. FIG. 10 shows the results thereof. As isapparent from the drawing, the compounds of the present invention can beefficiently used as labels.

EXAMPLE 10

An example of immunoassay using the compound of the present invention(Compound 4 shown in FIG. 5) is hereafter provided.

(1) Labeling of an Anti-Human CRP (C-Reactive Protein) Antibody withBiotin

The anti-human CRP antibody was labeled with biotin in accordance withthe process described in (1) of Example 7.

(2) Preparation of SA-labeled Form

In accordance with the process described in (3) of Example 7, anSA-labeled form of Compound 4 shown in FIG. 5 was prepared and europiumwas further added thereto.

(3) Coating of a Microtiter Plate with an Anti-Human CRP Antibody

In accordance with the process described in (4) of Example 7, amicrotiter plate on which the anti-human CRP antibody had beenimmobilized was prepared.

(4) Implementation of Immunoassay

A human CRP standard preparation was subjected to a 10-fold serialdilution with 1% BSA-containing physiological saline, and 50 μl thereofwas added to each well of the microtiter plate. After the plate wasshaken at 37° C. for 1 hour, the plate was washed with 0.05% Tween20-containing physiological saline. Thereafter, the biotin-labeledanti-human CRP antibody obtained in (1) was diluted to 1 μg/l with theaid of 1% BSA-containing physiological saline, and 50 μl thereof wasdispensed to each well. After the plate was shaken at 37° C. for 1 hour,the plate was washed with 0.05% Tween 20-containing physiologicalsaline. Thereafter, the europium-labeled SA prepared in (2) was dilutedwith 1% BSA-containing physiological saline, and 50 μl thereof wasfractioniated to each well.

After the plate was allowed to stand at room temperature for 30 minutes,the plate was washed with 0.05% Tween 20-containing physiologicalsaline. The microtiter plate was subjected to the measurement of theamount of light emission with a time-resolved assay apparatus.

FIG. 11 shows the results thereof. As is apparent from the drawing,Compound 4 shown in FIG. 5 can be efficiently used as a label inimmunoassay.

Industrial Applicability

The present invention provides (1) a novel compound that easily forms acomplex and (2) a novel compound that can be easily reacted with aprotein and the like. Further, the present invention enables theeffective use of a label in immunoassay, nucleic acid assay, and otherassay techniques.

1. A compound represented by formula (I):R—Y—(—X′-Phe-COCH₂COC_(n′)F_(2n′+1))_(m)  (I) wherein: R is selectedfrom the group consisting of hydrogen, alkyl, phenyl, and moieties ofthe formulae:

wherein: X is selected from the group consisting of halide, —OSO₃CH₃,—OSO₂F, —OSO₂CF₃, —SO₂C₄F₉, and

R^(A) is selected from the group consisting of alkyl, alkenyl, aryl, andaralkyl; R^(B) is selected from the group consisting of alkylene,arylene, and aralkylene; p is an integer from 0 to 5; q is an integerfrom 2 to 10; and n is an integer from 1 to 20; n′ is an integer from 1to 5; m is 1,2, or 3; in the case where m is 1: Phe is 1,4-phenylene; Yis selected from the group consisting of CH_(2,) a carbocyclic ring, anda heterocyclic ring; in the case where Y denotes CH₂, X′ is selectedfrom the group consisting of S, NH, OCH₂ CONH, and NHCO; and in the casewhere Y denotes a carbocyclic ring or a heterocyclic ring, X′ isselected from the group consisting of O, S, NH, CH₂, OCH₂, CONH, andNHCO; and in the case where m is 2 or 3: Phe is phenylene; Y is selectedfrom the group consisting of CH₂, a carbocyclic ring, and a heterocyclicring; X′ is selected from the group consisting of O, S, NH, CH₂, OCH₂,CONH, and NHCO.
 2. The compound of claim 1 represented by the formula:

wherein * denotes C_(n′)F_(2n′+1) (wherein n′ is an integer between 1and 5) and R is as defined in claim
 1. 3. The compound of claim 1represented by the formula:

wherein * denotes C_(n′)F_(2n′+1) (wherein n′ is an integer between 1and 5) and R is as defined in claim
 1. 4. The compound of claim 1represented by the formula:

wherein * denotes C_(n′)F_(2n′+1) (wherein n′ is an integer between 1and 5) and R is as defined in claim
 1. 5. The compound of claim 1represented by the formula:

wherein * denotes C_(n′)F_(2n′+1) (wherein n′ is an integer between 1and 5) and R is as defined in claim
 1. 6. The compound of claim 1represented by the formula:

wherein * denotes C_(n′)F_(2n′+1) (wherein n′ is an integer between 1and 5) and R is as defined in claim
 1. 7. The compound of claim 1represented by the formula:

wherein * denotes C_(n′)F_(2n′+1) (wherein n′ is an integer between 1and 5) and R is as defined in claim
 1. 8. The compound of claim 1represented by the formula:

wherein * denotes C_(n′)F_(2n′+1) (wherein n′ is an integer between 1and 5) and R is as defined in claim
 1. 9. The compound of claim 1represented by the formula:

wherein * denotes C_(n′)F_(2n′+1) (wherein n′ is an integer between 1and 5) and R is as defined in claim
 1. 10. The compound of claim 1represented by the formula:

wherein * denotes C_(n′)F_(2n′+1) (wherein n′ is an integer between 1and 5) and R is as defined in claim
 1. 11. The compound of claim 1represented by the formula:

wherein * denotes C_(n′)F_(2n′+1) (wherein n′ is an integer between 1and 5) and R is as defined in claim
 1. 12. The compound of claim 1represented by the formula:

wherein * denotes C_(n′)F_(2n′+1) (wherein n′ is an integer between 1and 5) and R is as defined in claim
 1. 13. The compound of claim 1represented by the formula:

wherein * denotes C_(n′)F_(2n′+1) (wherein n′ is an integer between 1and 5) and R is as defined in claim
 1. 14. A fluorescent complexcomprising the compound of claim 1 and a rare earth ion.
 15. A labelingreagent comprising the compound of claim 1 or the fluorescent complex ofclaim
 14. 16. The labeling reagent of claim 15 for immunoassays ornucleic acid assays.
 17. A method for labeling a protein, peptide, aminoacid, nucleic acid, or nucleotide utilizing the labeling reagent ofclaim 15.